The objective of this proposal is to characterize the receptors and signaling pathways that mediate initial and sustained MLC20 phosphorylation and contraction in smooth muscle of the gut by excitatory neurotransmitters (acetylcholine and opioid peptides), and by modulatory endocannabinoids. Our recent studies have shown that contraction induced by Gq/13-coupled receptors (m3, motilin, CCKA, S1P2, ETA) consists of a transient Ca2+-dependent phase mediated by MLC kinase, and a sustained, Ca2+-independent phase mediated by inhibition of MLC phosphatase (MLCP). Inhibition of MLCP is mediated by a RhoA-dependent pathway involving coordinated phosphorylation of two regulatory proteins, MYPT1 and CPI-17that inhibit MLCP and induce sustained MLC20 phosphorylation and contraction. Gi-coupled receptors (e.g.,d-opioid), however, activated PI3-kinase but not RhoA, implying operation of a distinct mechanism for inhibition of MLCP and sustained phosphorylation of MLC20 and contraction. Our preliminary studies indicate that the pathway involves PI3-kinase-dependent activation of integrin-linked kinase (ILK).-Cannabinoid CB1 receptors were coupled to an atypical G protein (Gai2/G[unreadable]5/C?-like RGS6) that precluded activation of downstream G[unreadable]?-dependent pathways. Accordingly, the specific aims of the proposal are: (1) to characterize d-opioid, m2, and CB1 receptor phosphorylation, desensitization, and internalization, and the roles of PKC-dependent RKIP (Raf-kinase inhibitory protein), Src-dependent caveolin-1, and GRK2/GRK5-dependent clathrin in these processes; (2) to characterize regulation and cross-regulation at G protein level by specific RGS proteins (RGS12 for m2 and d-opioid receptor-activated Gai3 and Gai2; RGS6 for CB1-activated Gai2; and RGS4 and RGS16 for m3-activated Gaq and Gai3); and (3) to characterize novel downstream pathways for inhibition of MLC phosphatase and phosphorylation of MLC20 by Gi-coupled receptors involving activation of ILK by PI3-kinase, and the mechanisms for inactivation of ILK and CPI-7via p38 MAP kinase-dependent PP2A and PP2C. Each specific aim is supported by substantial preliminary studies. These studies will provide a comprehensive analysis of novel signaling pathways initiated by prototypical Gi-coupled receptors in smooth muscle of the gut, and the interplay with signaling pathways initiated by Gq/13-coupled receptors. The studies will extend understanding of the function of Gi-coupled receptors in smooth muscle of the gut and in other tissues. [unreadable] [unreadable]